Overview

  • Product name

    Phospho-JAK1 (Y1022/Y0123) + Total In-Cell ELISA Kit
    See all JAK1 kits
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Human
  • Product overview

    Phospho-JAK1 (Y1022/Y1023) + Total In-Cell ELISA Kit (ab207475) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) JAK1 and/or total JAK1. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated JAK1 or total JAK1. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    The Phospho-JAK1 (Y1022/Y1023) + Total In-Cell ELISA Kit (ab207475) contains 96-well plates and two primary antibodies. The phospho-JAK1 antibody was raised against a synthetic phospho-peptide corresponding to residues surrounding Tyr1022 and Tyr1023 of human JAK1. This antibody recognizes JAK1 only when phosphorylated at these sites and does not cross-react with related kinases. The total-JAK1 antibody recognizes JAK1 proteins regardless of the phosphorylation state.


    The kit can be used to study phosphorylated JAK1 relative to cell number or to determine JAK1 phosphorylation relative to the total JAK1 protein found in the cells. Once the phospho-JAK1 and total-JAK1 signals have been normalized for cell number, a comparison of the ratio of phosphorylated JAK1 to total JAK1 for each of the cell growth conditions can be made.

  • Notes

    Members of the JAK family of tyrosine kinases (JAK1, JAK2, JAK3 and Tyk2) are phosphorylated on multiple tyrosine residues after stimulation by cytokines, and in turn serve as docking sites for other signal-transducing proteins containing SH2 domains. JAK kinases play critical roles in the pathogenesis of various human neoplastic diseases. They have been shown to modulate activation of Ras-Raf-MAP kinase pathways and are also involved in interferon-signaling. In addition, JAK kinases promote malignant cell survival and growth by modulating apoptosis via regulation of the Bcl-2 family members Bcl-XL, Bcl-2 and Bax. Bcl-2 regulation is independent from JAK kinase effects on STATs and other receptor linked pathways such as the PI3K and the Ras-MAPK.

    JAK1 (Janus Kinase 1) is a 130 kDa non-receptor protein tyrosine kinase. JAK1 is a widely expressed, membrane-associated kinase involved in many signal transduction pathways including those involving interferon α, β and γ, all class II receptors and cytokine receptors that utilize the common γ chain or gp130 subunit. JAK1 can be activated via phosphorylation on tyrosines 1022 and 1023 in the kinase activation loop. Once activated, JAK1 phosphorylates STAT proteins and SHP2. Phosphorylated STATs then dimerize and translocate to the nucleus. Once in the nucleus, STAT proteins bind to DNA and modify the transcription of various genes, which leads to various responses such as cell proliferation, cell survival and differentiation.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    1% SDS Solution 1 x 22ml 5 x 22ml
    10% Triton X-100 1 x 10ml 5 x 10ml
    10X PBS 1 x 120ml 5 x 120ml
    1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml
    1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml
    96-well tissue culture plate 2 units 10 units
    Anti-rabbit HRP-conjugated IgG 2 x 11µl 10 x 11µl
    Crystal Violet Solution 1 x 22ml 5 x 22ml
    Developing Solution 2 x 11ml 10 x 11ml
    Phospho-JAK1 antibody 1 x 9µl 5 x 9µl
    Plate sealer 2 units 10 units
    Stop Solution 2 x 11ml 10 x 11ml
    Total-JAK1 antibody 1 x 9µl 5 x 9µl
  • Research areas

  • Function

    Tyrosine kinase of the non-receptor type, involved in the IFN-alpha/beta/gamma signal pathway. Kinase partner for the interleukin (IL)-2 receptor.
  • Tissue specificity

    Expressed at higher levels in primary colon tumors than in normal colon tissue. The expression level in metastatic colon tumors is comparable to the expression level in normal colon tissue.
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. JAK subfamily.
    Contains 1 FERM domain.
    Contains 1 protein kinase domain.
    Contains 1 SH2 domain.
  • Domain

    Possesses two phosphotransferase domains. The second one probably contains the catalytic domain (By similarity), while the presence of slight differences suggest a different role for domain 1.
    The FERM domain mediates interaction with JAKMIP1.
  • Cellular localization

    Endomembrane system. Wholly intracellular, possibly membrane associated.
  • Information by UniProt
  • Alternative names

    • JAK 1
    • JAK 1A
    • JAK 1B
    • JAK-1
    • JAK1
    • JAK1_HUMAN
    • JAK1A
    • JAK1B
    • Janus kinase 1
    • Janus kinase 1 (a protein tyrosine kinase)
    • JTK3
    • Tyrosine protein kinase JAK 1
    • Tyrosine protein kinase JAK1
    • Tyrosine-protein kinase JAK1
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207475 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

Images

  • Measurement of phosphorylated and total JAK1. 3T3-L1 cells were cultured in 96-well plates for 48 hours and converted to adipocytes. Cells were then serum-starved for 16 hours, treated with 20 ng/ml LIF (Leukemia Inhibitory Factor) for 10 minutes and fixed. Total and phospho-JAK1 were each assayed in triplicate using the phospho-JAK and total JAK1 antibodies included in the kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Protocols

References

ab207475 has not yet been referenced specifically in any publications.

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